Meta-Analysis of Assessment of Total Oxidative Stress and Total Antioxidant Capacity in Patients with Periodontitis

Background Periodontitis is intricately linked to oxidative stress-antioxidant (redox) imbalance. The antioxidant system scavenges the oxygen free radicals in biological fluids in patients with periodontitis. However, little is still known about the free radicals mediated oxidative stress and reductive ability of the antioxidant system. Thus, the present meta-analysis aims to quantitatively review the literature that assessed the oxidative stress marker total oxidative stress (TOS) and total antioxidant capacity (TAC) in various biological fluids of patients with periodontitis. Methodology. Electronic databases were searched for studies that assessed TOS and TAC levels in various biological samples of patients with periodontitis. Results From the 1,812 articles identified, 1,754 were excluded based on title and abstract screening due to irrelevance to the topic of interest. A full-text assessment of the remaining 58 articles led to the selection of 42 articles that satisfied the inclusion criteria. Of these, only 24 studies had consistent data for quantitative analysis. The periodontitis group displayed significantly elevated TOS levels (p < 0.05) in serum, gingival crevicular fluid (GCF), and saliva samples in the studies evaluated. In contrast, the periodontitis group exhibited significantly attenuated TAC levels (p < 0.01) compared to healthy controls in plasma, serum, and GCF samples of the studies evaluated, which was insignificant in salivary samples (p=0.433). At the same time, the periodontitis group displayed insignificantly elevated TAC levels after periodontal therapy (p=0.130). Conclusions The present meta-analysis showed significantly higher TOS and lower TAC in periodontitis, reflecting the elevated oxidative stress level than the control group. Clinical Relevance. Scientific rationale for the study: The imbalance between oxidants and antioxidants (oxidative stress (OS)) plays a critical role in the onset and progression of periodontitis; the assessment of the relationship between OS-related biomarkers in regional samples and systemic samples of patients with periodontitis helps us to evaluate the periodontal disease progression. The OS biomarker levels can be used to assess periodontal disease and therapeutic efficacy.


Introduction
Reactive oxygen species or free radicals are anions with one unpaired electron in the outer shell.The complex effect of enzymatic and nonenzymatic antioxidants scavenge or neutralize the free radical activity [1].Indeed, when the antioxidant reserve system is exhausted by the excessive accumulation of free radicals, there is no scavenging of reactive oxygen species (ROS) nor their neutralization.The resultant disruption of the antioxidant barrier is directly responsible for oxidative stress (OS)/nitrosative stress-mediated modifications of biological components [2].OS plays a primary role in the etiopathogenesis of many systemic diseases, such as diabetes and cardiovascular diseases, as well as a possible risk factor for chronic renal failure, rheumatoid arthritis, and neurodegenerative diseases [3].Chronic inflammatory disease, like periodontitis, is also intricately associated with oxidative-reductive imbalance.Many studies have shown that OS is directly responsible for the progressive degradation of extracellular matrix components of the periodontal attachment apparatus [4].Recent literature indicates that redox disturbances are intensified in periodontitis patients with comorbidities.Therefore, the present meta-analysis aims to assess the literature that evaluated the total oxidative and antioxidative capacity of various biological fluids in patients with periodontitis.

Materials and Methods
2.1.Registration of the Protocol.PRISMA guidelines have been strictly followed for study selection.The meta-analysis protocol was recorded in the PROSPERO database (CRD42021281819).
2.2.Question of Observation.Is there any significant difference in the total oxidative stress (TOS) and total antioxidant capacity (TAC) levels of biological fluid samples between patients with periodontitis and the healthy control group?
Based on the research question, the following components were formulated: (i) Patient population: patients with periodontitis (ii) Exposure or marker of evaluation: mean and standard deviation value of TOS and TAC values (iii) Comparison: between patients with periodontitis and healthy control group (iv) Outcome: assessment of TOS and TAC in various biological fluid samples of patients with Periodontitis (v) Study: collect the literature of cross-sectional and case-controlled studies that evaluated the status of TOS and TAC in Periodontitis and control from 2000 to 2023.
2.3.Literature Search.Electronic databases, including PubMed, ScienceDirect, Cochrane, Wiley Online Library, and Cross Reference, were searched for published articles addressing the TOS and TAC in patients with periodontitis and the control group between 2000 and 2023.The keywords were employed: "periodontal disease," "total oxidative stress," "oxidative stress," and "total antioxidant capacity." 2.4.Screening for Selection.Articles discussing oxidative stress and TAC in periodontitis were collected and screened for relevance based on the titles and abstracts.
2.5.Inclusion Criteria.Studies discussed the TOS and TAC in patients with periodontitis and the healthy control group; (i) Both the case and control groups consisted of individuals who were in good systemic health, refraining from the use of antibiotics, anti-inflammatories, or any other medications.Moreover, they had not undergone any periodontal treatments within the past 3 months.
(ii) The inclusion of studies examining systemic diseases in relation to periodontitis was contingent on the presence of two distinct evaluation groups: one for systemically healthy controls and another for the periodontitis-afflicted group.(iii) Studies investigating the correlation between periodontitis and smoking were considered only if they incorporated nonsmokers in both the periodontitis group and the control group as separate evaluation groups.(iv) Studies examining the impact of therapy on periodontitis were included only when they provided baseline values for both the periodontitis group and the control group.
Studies involving various biological fluid samples and expressed the assessed TOS and TAC values in mean, standard deviation, along with probability value: (i) Papers provided consistent data to allow comparison of patient and control groups with other relevant studies.
2.6.Exclusion Criteria.Articles with the unmatched title and objectives: (i) Studies that were duplicates, when they involved the same subjects and by the same authors.(ii) Observational studies exclusively focusing on pregnant women or children.(iii) The studies examined the therapeutic effect but did not include an evaluation of the control group.(iv) Studies included patients with systemic diseases or smokers but did not incorporate separate evaluation groups for systemically healthy individuals or nonsmokers in the periodontitis and control groups.2.7.Full-Text Retrieval and Evaluation.Two authors screened the titles and objectives of the collected studies and excluded the presentations at higher risk of bias from the quantitative synthesis based on predefined criteria.Two authors have independently evaluated the full text of each included study.Two authors have collected data for the factors considered in the meta-analysis.After considering all the particulars, the authors have selected the articles for eligibility criteria.The authors resolved disagreements by 2 Disease Markers consensus.Finally, all the authors participated in manuscript preparation.
2.8.Data Segregation.The extracted information from the full text of selected articles as the author, year of publication, sample size, TOS and TAC measurements in Periodontitis, and control group expressed as the mean with standard deviation along with specific measurement units and assessment methods.
2.9.Statistical Analysis.The meta-analysis was performed by deriving the forest plot using the standard mean difference method using comprehensive meta-analysis software version 3 (Biostat Inc.Englewood, NJ, USA).The standardized mean difference values of TOS and TAC in periodontitis were evaluated at a 95% confidence interval (CI).Due to significant heterogeneity, a random-effects model was chosen for the analysis.The studies that expressed the TOS and TAC levels in similar units in each sample were only selected for the meta-analysis.

Results
PubMed search yielded 783 papers, ScienceDirect search yielded 369 papers, Wiley Online Library yielded 265 papers, Cochrane search yielded 390 papers, and Cross-reference search yielded five papers.After search refinement, 1,754 articles were excluded due to unmatched titles and abstracts, including four duplicated data reports and one animal study.
After the extraction of these articles, 58 articles had titles relevant to the present work.The full text was retrieved for the screened articles.Papers not meeting the selection criteria (n = 16) were excluded.Finally, 42 articles with matched objectives were selected for the systematic review.Only 24 articles had data compatible with the meta-analysis (Figure 1).Newcastle-Ottawa scale of included studies in the metaanalysis was displayed in Table 1 .Collected TOS and TAC assessment data, criteria for periodontitis diagnosis, and other relevant findings from included articles in various Disease Markers biological fluid samples were tabulated in Tables 2 and 3, respectively [47][48][49][50][51][52].The different methodologies utilized for assessing TOS and TAC values were also displayed in Tables 2 and 3 [53][54][55][56][57][58][59][60][61].The pre and posttreatment mean values of TAC in different biological fluid samples in patients with periodontitis data from the included studies are presented in Table 4.The analysis of TOS levels after therapeutic intervention could not be performed due to the scarcity of published studies.
Selection-Case definition, case selection, control definition, and selection.
Comparability-Consideration of matching known and potential confounding factors.
Exposure-Securing patient records, interviewer blindness to groups, similarity ascertainment between groups, and nonresponse rate.
Each criterion was awarded with one star.The overall score was determined by summing the awarded stars.Studies The method utilized for the assessment of TOS values in various biological samples was Erel [53].SD, standard deviation; Cont, control; H

8
Disease Markers receiving scores in the range of 6-9 were categorized as highquality, those scoring between 3 and 5 were classified as fairquality, and studies with scores ranging from 0 to 2 were deemed to be of poor quality.Importantly, it's worth noting that all the studies analyzed received scores exceeding 6, indicating a minimal risk of bias.
The periodontitis group displayed significantly elevated TOS levels (p<0:05) in serum, gingival crevicular fluid (GCF), and saliva samples in the studies evaluated.In contrast, the periodontitis group exhibited significantly attenuated TAC levels (p<0:01) compared to healthy controls in serum, plasma, and GCF samples of the studies evaluated, which was insignificant in salivary samples (p ¼ 0:433).At the same time, the periodontitis group displayed insignificantly elevated TAC levels after periodontal therapy (p ¼ 0:130).
The GCF samples showed an overall mean difference TAC value of −2.004 mmol Trolox equivalent (TEq)/l with 95% CI (−3.490 to −0.517) (Figure 5).The salivary samples  The forest plot shows standard mean difference estimates with 95% confidence intervals representing differences in GCF levels of TOS between the patients with periodontitis and healthy controls.
Disease Markers displayed an overall standard mean difference TAC value of −0.709 mmol TEq/l with 95% CI (−2.481 to 1.063) (Figure 6).The serum samples showed an overall standard mean difference TAC value of −2.049 mmol TEq/l with 95% CI (−3.018 to −1.079) (Figure 7).The plasma samples showed an overall standard mean difference TAC value of −0.959 µmol TEq/l with 95% CI (−1.504 to −0.415) (Figure 8).The overall standard mean difference TAC value pre and posttherapy is 0.666 µmol TEq/l with 95% CI (−0.196 to 1.528) (Figure 9).The meta-analysis of the TOS assessment presented high heterogeneity, reflected by the I 2 values 87.256, 85.382, and 83.513 in Figure 2-4, respectively.The meta-analysis of the TAC assessment presented high heterogeneity, reflected by the I 2 values 95.494, 98.970, 94.749, 82.757, and 88.617 in Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9, respectively.The different methodologies utilized to measure TOS and TAC values could cause high heterogeneity.
3.1.Publication Bias.Studies included in this assessment of TOS in periodontitis meta-analysis showed Egger's regression intercept values 9.162, −6.856, 3.689 with two-tailed p-values 0.45, 0.45, and 0.701 in saliva, GCF, and serum samples, respectively, indicating a low risk of publication bias of included studies in meta-analysis.Studies included in this assessment of TAC in periodontitis meta-analysis showed Egger's regression intercept values −12.79, 1.895, −23.06 with two-tailed p-values 0.48, 0.67, and 0.07 in GCF, plasma, and saliva samples, respectively, denoting a lower risk of publication bias of included studies in meta-analysis.Studies included in this assessment of serum TAC in periodontitis meta-analysis showed Kendall's S statistic (P-Q) value of −20.0 with two-tailed p-values of 0.051 (Begg and Mazumdar's test for rank correlation), denoting a moderate risk of publication bias of included studies in the salivary assessment of TAC meta-analysis.The studies included the posttherapy TAC assessment in periodontitis meta-analysis showed Eggers regression intercept value of −2.67 with a two-tailed p-value of 0.73, indicating a low risk of publication bias of included studies in meta-analysis.

Discussion
Despite the increasing knowledge of the etiopathogenesis of inflammatory periodontal diseases, there are no definitive indicators for objectivizing the diagnosis, determining the disease's severity, and evaluating treatment results.Hence, the present meta-analysis assessed the literature evaluated the oxidative stress markers such as TOS and TAC levels in the blood, GCF, and both stimulated and nonstimulated saliva in patients with periodontitis to find the validity of these markers in determining the diagnosis and prognosis of periodontitis as well as the treatment effects.In the present meta-analysis, Out of 10 studies of TOS assessment in serum, GCF, and saliva involving 260 patients in the study group and 212 in the control group, seven studies proved a significantly higher TOS in patients with periodontitis [9,16,19,[26][27][28]42].They also reported a strong correlation of TOS with the clinical parameters of periodontitis [12,16].This fact is unsurprising since the stimulated saliva secreted by the parotid gland is the primary source of free radicals (ROS) [62,63].However, Zhang et al. [64] showed no difference in the salivary TOS levels between periodontitis and healthy controls.Further, they found that a high bacterial load did not depict any correlation with salivary TOS values [64].Toczewska et al. [65] found a weak correlation of TOS values with clinical periodontal parameters.These differences might be due to the selection criteria of patients in different studies.Since other factors related to subjects such as age, smoking, gender, and nutrition would yield distinct effects on the alteration of OS parameters; thus, it is essential to consider these factors when considering this parameter as a The forest plot depicts standard mean difference estimates with 95% confidence intervals representing differences in salivary levels of TOS between patients with periodontitis and healthy controls.
10 Disease Markers potential marker of periodontitis.Therefore, patient selection could be a crucial parameter influencing salivary levels of TOS.The present meta-analysis of the evaluated studies displayed significantly elevated TOS levels (p<0:05) in GCF, saliva, and serum samples of the periodontitis group with the overall standardized mean difference value of 1.011, 1.784, and 0.694 µmol H 2 O 2 Eq/l, respectively.
Out of 39 studies of TAC assessment in plasma, serum, GCF, and saliva, involving 1,418 patients in the study group and 1,340 in the control group, except 11 studies, the remaining 31 studies proved a significant decrease in the TAC in patients with periodontitis when compared to the clinically healthy periodontium [5, 6, 8, 14, 21-24, 27, 38, 39].Chapple et al. [8] reported that GCF/salivary TAC values were significantly higher in patients with periodontitis than in the control group.The authors attributed that the initial increase in antioxidant response in periodontitis is due to local reactive or adaptive response to a first phase increase of oxidative burst (OS) occurring with periodontal inflammation.The adaptive antioxidant defense might decrease over time as ROS production becomes chronic.The excess utilization of antioxidants to neutralize the exaggerated ROS activity during periodontal inflammation results in the depletion of TAC  Disease Markers 13 reported that neither gingivitis nor smoking habits influence salivary TAC values [72].The variations in the results of TAC between different studies could be due to different analytical methods utilized in assessing TAC values.The present meta-analysis of the evaluated studies exhibited significantly attenuated TAC levels (p<0:01) in the periodontitis group compared to healthy controls in GCF, serum, and plasma samples, which was insignificant in salivary samples (p ¼ 0:433).The overall mean difference of TAC value in GCF, salivary, serum, and plasma samples were −2.004, −0.709, −2.049, and −0.959 µmol TEq/l.
At the same time, the periodontitis group displayed insignificantly (p ¼ 0:130) elevated TAC levels after periodontal therapy.The overall standard mean difference TAC value upon comparison of pre and posttherapy was 0.666 µmol TEq/l.
Although high heterogeneity was detected among included studies of the present meta-analysis, our results still indicate that periodontitis has statistically correlated with some local OS biomarkers, and oxidative stress played a critical role in the pathological process of periodontitis.Thus, TOS and TAC may be helpful and practical biomarkers for evaluating oxidative injury in periodontal tissues.

Conclusions
In conclusion, the present meta-analysis supports the rationale that there is a direct link between periodontitis and OSrelated biomarkers in the local site.The imbalance of ROS and antioxidant systems may contribute to functional and structural remodeling that favors the occurrence of periodontitis.Furthermore, these two measurements can potentially evaluate the interaction between periodontal and systemic status and the effectiveness of periodontal treatment.Considering the above facts, it may be speculated that oxidative stress is an essential factor in periodontitis.Studying the antioxidant defense mechanisms may be regarded as a valuable biomarker that will help better understand the underlying pathology of tissue damage and novel therapeutic interventional strategies.Therefore, it is necessary to conduct further research on a larger number of patients with periodontitis to understand the oxidative stress and antioxidant status imbalance reported in patients with periodontitis.
(v) Being literature, critical, or systematic reviews.(vi) Studies used other markers of oxidative stress and antioxidant capacity for evaluation.(vii) The articles provided incompatible data for the comparison between control and periodontitis groups with other studies.(viii) The studies expressed the results in Graphical representation without the accurate value display.(ix) Studies discussed the TOS and TAC values in other oral chronic inflammatory diseases.

FIGURE 2 :
FIGURE 2:The forest plot shows standard mean difference estimates with 95% confidence intervals representing differences in GCF levels of TOS between the patients with periodontitis and healthy controls.

FIGURE 9 :
FIGURE 9: Meta-analysis of TAC levels between before and after treatment of periodontitis group.

TABLE 1 :
Newcastle-Ottawa Scale for studies included in the meta-analysis.

TABLE 2 :
The mean values of TOS in various biological fluid samples between healthy controls and patients with periodontitis in the included studies of quantitative synthesis.

TABLE 3 :
The mean values of TAC in various biological fluid samples between healthy controls and patients with periodontitis in the included studies included in the meta-analysis.

TABLE 4 :
The pre and posttreatment mean values of TAC in various biological fluid samples in patients with periodontitis in the included studies of quantitative synthesis.
The treatment performed was non-surgical therapy (NST).SD, standard deviation.Forest plot shows GCF levels of TOS between the patients with periodontitis and healthy control groups Forest plot shows the levels of TOS in saliva between patients with periodontitis and healthy controls Forest plot shows TOS values of serum between periodontitis and control The forest plot displays standard mean difference estimates with 95% confidence intervals representing differences in serum levels of TOS between patients with periodontitis and healthy controls.The forest plot shows standard mean difference values of TAC in GCF samples with 95% confidence intervals between patients with the periodontitis group and healthy controls.The forest plot shows salivary TAC standard mean difference values with 95% confidence intervals between patients with the periodontitis group and healthy controls.The forest plot shows plasma TAC standard mean difference values with 95% confidence intervals between patients with periodontitis and healthy controls.Forest plot shows comparison of pre and posttreatment TAC values between periodontitis and control group [6,32,71]anjamurthy et al.[70]reported higher serum, GCF, and salivary TAC values in periodontitis participants.However, some studies also showed that salivary TAC increases or remains at the same level in periodontitis patients compared to healthy controls[6,32,71].Another study